ABSTRACT The proposed multidisciplinary research will focus on the development of a novel pharmacological intervention that employs 10?,17?-dihydroxyestra-1,4-dien-3-one (DHED, a bioprecursor prodrug of estradiol that exerts no systemic side-effects) to control neurodegeneration of retinal ganglion cells in glaucoma after topical administration. To this end, preclinical testing of the new therapeutic strategy will be performed, along with in vitro ocular biotransformation, in vivo distribution and proteomics studies, in models of human glaucoma. These experiments will determine efficacy of DHED treatment in terminating and/or preventing glaucoma- associated neuronal loss, and to generate data to support feasibility of the proposed intervention. Specifically, we will test the two-pronged hypothesis that treatment with the prodrug DHED elicits protection of retinal ganglion cells upon topical delivery in established models of human glaucoma and, therefore, leads to prevention or improvement of visual impairment and functional deficits associated with glaucoma. The hypothesis will be tested with specific aims to 1) evaluate of metabolic and transport properties of DHED in the eye, as well as to determine of the distribution of DHED and estradiol after topical administration; 2), and to perform proteomics in experimental animals with focus on therapeutic target engagement in the retina; 3) meausure pharmacological parameters of retinal ganglion cell protection by treatment with DHED in an in vitro model; 4) determine therapeutic parameters in the neuroprotection of retinal ganglion cells in an in vivo animal model of human glaucoma and to identify the effect of protection of retinal ganglion cells on improvement of visual function in the animal model due to treatment with DHED. The determination of neuronal viability and the acquired knowledge on associated biopharmaceutical and pharmacological parameters will indicate the potential of the method to remedy glaucoma as the long-term goal of the project. This non-intraocular pressure lowering approach will have the prospective to be both preventative and therapeutic in nature and to complement existing glaucoma treatment designs and rationales. A successful completion of the project will document critical elements in assessing the potential of DHED approach to be an effective site-specific delivery agent of E2 for retinal neuroprotection, and the proposed experiments will likely lay the groundwork for the further development of novel intervention to combat devastating ophthalmic neurodegenerative diseases.